U.S. patent application number 13/521622 was filed with the patent office on 2013-05-30 for methods of using fut2 genetic variants to diagnose crohn's disease.
This patent application is currently assigned to CEDARS-SINAI MEDICAL CENTER. The applicant listed for this patent is Xiuqing Guo, Dermot P. McGovern, Jerome I. Rotter, Stephan R. Targan, Kent D. Taylor. Invention is credited to Xiuqing Guo, Dermot P. McGovern, Jerome I. Rotter, Stephan R. Targan, Kent D. Taylor.
Application Number | 20130136720 13/521622 |
Document ID | / |
Family ID | 44304678 |
Filed Date | 2013-05-30 |
United States Patent
Application |
20130136720 |
Kind Code |
A1 |
McGovern; Dermot P. ; et
al. |
May 30, 2013 |
METHODS OF USING FUT2 GENETIC VARIANTS TO DIAGNOSE CROHN'S
DISEASE
Abstract
The present invention relates to prognosing, diagnosing and
treating of Crohn's disease. The invention also provides prognosis,
diagnosis, and treatment that are based upon the presence of one or
more genetic risk factors at the FUT2 genetic locus.
Inventors: |
McGovern; Dermot P.; (Los
Angeles, CA) ; Rotter; Jerome I.; (Los Angeles,
CA) ; Targan; Stephan R.; (Santa Monica, CA) ;
Taylor; Kent D.; (Ventura, CA) ; Guo; Xiuqing;
(Santa Monica, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
McGovern; Dermot P.
Rotter; Jerome I.
Targan; Stephan R.
Taylor; Kent D.
Guo; Xiuqing |
Los Angeles
Los Angeles
Santa Monica
Ventura
Santa Monica |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Assignee: |
CEDARS-SINAI MEDICAL CENTER
Los Angeles
CA
|
Family ID: |
44304678 |
Appl. No.: |
13/521622 |
Filed: |
January 14, 2011 |
PCT Filed: |
January 14, 2011 |
PCT NO: |
PCT/US11/21382 |
371 Date: |
July 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61295309 |
Jan 15, 2010 |
|
|
|
Current U.S.
Class: |
424/93.4 ;
506/2 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/156 20130101; C12Q 2600/172 20130101 |
Class at
Publication: |
424/93.4 ;
506/2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under NCRR
grant MOI-RR00425 and NIH grant P01-DK046763. The government has
certain rights in the invention.
Claims
1. A method of diagnosing susceptibility to Crohn's disease in an
individual, comprising: obtaining a sample from the individual;
assaying the sample to determine the presence or absence of a risk
variant at the FUT2 genetic locus; and diagnosing susceptibility to
Crohn's disease in the individual based on the presence of the risk
variant at the FUT2 genetic locus.
2. The method according to claim 1, wherein the risk variant is
selected from the group consisting of rs602662, rs676388, rs485186,
and rs504963.
3. The method of claim 1, wherein assaying the sample comprises
genotyping for one or more single nucleotide polymorphisms.
4. The method according to claim 1, wherein the sample is whole
blood, plasma, serum, saliva, cheek swab, urine, or stool.
5. A method of prognosing Crohn's disease in an individual,
comprising: obtaining a sample from the individual; assaying the
sample for the presence or absence of one or more genetic risk
variants; and prognosing an aggressive form of Crohn's disease
based on the presence of one or more risk variants at the FUT2
genetic locus.
6. The method according to claim 5, wherein the risk variant is
selected from the group consisting of rs602662, rs676388, rs485186,
and rs504963.
7. The method of claim 5, wherein assaying the sample comprises
genotyping for one or more single nucleotide polymorphisms.
8. The method according to claim 5, wherein the sample is whole
blood, plasma, serum, saliva, cheek swab, urine, or stool.
9. A method of treating an individual for Crohn's disease,
comprising: prognosing an aggressive form of Crohn's disease in the
individual based on the presence of one or more risk variants at
the FUT2 genetic locus; and treating the individual, wherein the
one or more risk variants are selected from rs602662, rs676388,
rs485186, and rs504963.
10. The method of claim 9, wherein assaying the sample comprises
genotyping for one or more single nucleotide polymorphisms.
11. The method according to claim 9, wherein the sample is whole
blood, plasma, serum, saliva, cheek swab, urine, or stool.
12. A method of determining a high probability of developing
Crohn's disease in an individual, relative to a healthy subject,
comprising: obtaining a sample from the individual; assaying the
sample to determine the presence or absence of one or more risk
variants at the FUT2 genetic locus; and diagnosing a high
probability of developing Crohn's disease in the individual,
relative to a healthy subject, based upon the presence of the one
or more risk variants at the FUT2 genetic locus.
13. The method according to claim 12, wherein the one or more risk
variants are selected from the group consisting of rs602662,
rs676388, rs485186, and rs504963.
14. The method of claim 12, wherein assaying the sample comprises
genotyping for one or more single nucleotide polymorphisms.
15. The method according to claim 12, wherein the sample is whole
blood, plasma, serum, saliva, cheek swab, urine, or stool.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of the filing
date of U.S. Provisional Application No. 61/295,309 filed Jan. 15,
2010, the disclosure of which is incorporated herein by reference
in its entirety.
FIELD OF INVENTION
[0003] The invention relates generally to the field of inflammatory
disease, specifically to Crohn's disease.
BACKGROUND
[0004] Crohn's disease (CD) and ulcerative colitis (UC), the two
common forms of idiopathic inflammatory bowel disease (IBD), are
chronic, relapsing inflammatory disorders of the gastrointestinal
tract. Each has a peak age of onset in the second to fourth decades
of life and prevalences in European ancestry populations that
average approximately 100-150 per 100,000 (61, 62). Although the
precise etiology of IBD remains to be elucidated, a widely accepted
hypothesis is that ubiquitous, commensal intestinal bacteria
trigger an inappropriate, overactive, and ongoing mucosal immune
response that mediates intestinal tissue damage in genetically
susceptible individuals (62). Genetic factors play an important
role in IBD pathogenesis, as evidenced by the increased rates of
IBD in Ashkenazi Jews, familial aggregation of IBD, and increased
concordance for IBD in monozygotic compared to dizygotic twin pairs
(63). Moreover, genetic analyses have linked IBD to specific
genetic variants, especially CARD15 variants on chromosome 16q12
and the IBD5 haplotype (spanning the organic cation transporters,
SLC22A4 and SLC22A5, and other genes) on chromosome 5q31 (12, 63,
64, 65, 66). CD and UC are thought to be related disorders that
share some genetic susceptibility loci but differ at others.
[0005] The replicated associations between CD and variants in
CARD15 and the IBD5 haplotype do not fully explain the genetic risk
for CD. Thus, there is need in the art to determine other genes,
allelic variants and/or haplotypes that may assist in explaining
the genetic risk, diagnosing, and/or predicting susceptibility for
or protection against inflammatory bowel disease including but not
limited to CD and/or UC.
SUMMARY OF THE INVENTION
[0006] In one embodiment, the invention provides a method of
diagnosing susceptibility to Crohn's disease in an individual,
comprising: obtaining a sample from the individual, assaying the
sample to determine the presence or absence of a risk variant at
the FUT2 genetic locus, and diagnosing susceptibility to Crohn's
disease in the individual based on the presence of the risk variant
at the FUT2 genetic locus. The risk variant can be selected from
the group consisting of rs602662, rs676388, rs485186, and rs504963.
Assaying of the sample comprises genotyping for one or more single
nucleotide polymorphisms. The sample can be whole blood, plasma,
serum, saliva, cheek swab, urine, or stool.
[0007] In another embodiment, the invention provides a method of
determining a high probability of developing Crohn's disease in an
individual, relative to a healthy subject, comprising: obtaining a
sample from the individual, assaying the sample to determine the
presence or absence of one or more risk variants at the FUT2
genetic locus, and diagnosing a high probability of developing
Crohn's disease in the individual, relative to a healthy subject,
based upon the presence of one or more risk variants at the FUT2
genetic locus. The risk variant can be selected from the group
consisting of rs602662, rs676388, rs485186, and rs504963 Assaying
of the sample comprises genotyping for one or more single
nucleotide polymorphisms. The sample can be whole blood, plasma,
serum, saliva, cheek swab, urine, or stool.
[0008] In a related embodiment, the invention provides a method of
prognosing Crohn's disease in an individual, comprising: obtaining
a sample from the individual, assaying the sample for the presence
or absence of one or more genetic risk variants, and prognosing an
aggressive form of Crohn's disease based on the presence of one or
more risk variants at the FUT2 genetic locus. The risk variant can
be selected from the group consisting of rs602662, rs676388,
rs485186, and rs504963. Assaying of the sample comprises genotyping
for one or more single nucleotide polymorphisms. The sample can be
whole blood, plasma, serum, saliva, cheek swab, urine, or
stool.
[0009] In a further embodiment, the invention provides method of
treating an individual for Crohn's disease, comprising: prognosing
an aggressive form of Crohn's disease in the individual based on
the presence of one or more risk variants at the FUT2 genetic
locus, and treating the individual, wherein the one or more risk
variants are selected from rs602662, rs676388, rs485186, and
rs504963. Assaying the sample comprises genotyping for one or more
single nucleotide polymorphisms. The sample can be whole blood,
plasma, serum, saliva, cheek swab, urine, or stool.
[0010] The above-mentioned and other features of this invention and
the manner of obtaining and using them will become more apparent,
and will be best understood, by reference to the following
description, taken in conjunction with the accompanying drawings.
The drawings depict only typical embodiments of the invention and
do not therefore limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
[0011] Exemplary embodiments are illustrated in referenced figures.
It is intended that the embodiments and figures disclosed herein
are to be considered illustrative rather than restrictive.
[0012] FIG. 1. Graphical representation of an association between
FUT2and CD. Circles--The GWAS population. Squares--The independent
case-control replication cohort.
[0013] FIG. 2. Principal Component Plot for components 1 (C1--y
axis) and 2 (C2--x axis) in CD and controls. The circled cases and
controls are on the `Caucasian` axis and were included in logistic
regression analysis.
[0014] FIG. 3. Table listing the replication of confirmed and
`nominally associated` CD susceptibility loci from CD GWAS
meta-analysis.sup.13. Chr.--chromosome
[0015] FIG. 4. Table listing novel loci associated with CD (cut off
p=<1.0.times.10.sup.4). Chr.--chromosome.
[0016] FIG. 5. Table summarizing the association between FUT2 and
CD in GWAS, confirmatory cohort of 1174 cases and 357 controls and
the p value for association by the CD GWAS meta-analysis from
Barrett et al. *P value calculated using logistic regression.
**Combined p value calculated for p value in original GWAS and one
tailed p value in independent replication. Synon.--synonymous.
[0017] FIG. 6. Graphical representation of the linkage
disequilibrium and haplotype structure across the 6 FUT2 SNPs.
Figure and data generated in HAPLOVIEW. Figures represent the LD in
percent between SNPs as represented by D'.
DESCRIPTION OF THE INVENTION
[0018] Crohn's disease (CD), one of the major forms inflammatory
bowel diseases (IBD), is a chronic, debilitating disease
characterized by recurrent gastrointestinal inflammation,
postulated to occur as a result of an abnormal immune reaction to
commensal flora in genetically susceptible individuals. The role of
commensal flora in potentiating chronic gastrointestinal mucosal
inflammation is substantiated by data from established rodent
models of IBD such as the Il10.sup.-/- mouse and the Hla-B27
transgenic rat that are disease free when kept in germ free
environments but develop inflammation when raised under pathogen
free conditions (1,2) Furthermore, in both of these models, the
bacterial load and the nature of the commensal flora can influence
either the site or degree of gastrointestinal inflammation (1,3,4).
In human disease, antibiotic and probiotic therapy can be effective
in modifying some of the manifestations of IBD (5,6).
[0019] Through utilizing genome-wide association studies (GWAS), in
addition to candidate gene approaches, considerable success has
been achieved in identifying genetic loci that increase
susceptibility to CD in populations of Northern European origin
(7-12). To date more than thirty loci are definitively known to be
associated with CD, although these loci only account for a minority
of the genetic variance to CD in the Caucasian population (13). A
number of the CD susceptibility genes encode important components
of the innate immune system genes such as NOD2 (11,12). The Toll
like receptors (14,15) and the autophagy genes ATG16L1 and IRGM,
emphasizing the importance of the microbial-host interaction in the
development of CD. Furthermore, antibodies to bacterial antigens
have been identified that define certain sub-groups of CD patients,
reinforcing the essential role that bacteria play in driving CD
(16).
[0020] As disclosed herein, a CD genome-wide association study
(GWAS) was performed by the inventors, identifying a number of
novel associations with CD. Considering the importance of the
host-microbial interaction, the novel association with
Fucosyltransferase 2 (FUT2), also termed secretor factor (Se), was
of particular interest. FUT2 is a physiological trait that
regulates the expression of the H antigen, a precursor of the blood
group A and B antigens, on the gastrointestinal mucosa.
Approximately 20% of Caucasians are non-secretors who do not
express ABO antigens in saliva as they are homozygous for FUT2 null
alleles (17). Genetic variation in FUT2 has been implicated in
susceptibility to Helicobacter pylori infection (18), Noroviruses
(Norwalk virus) (19-21), and progression of HIV (22). FUT2 alleles
have also been associated with circulating serum vitamin B12 levels
(23). Furthermore non-secretion of ABO blood group antigens into
body fluids has been shown to be associated with the development of
oral candidiasis (24,25), rheumatic fever (26), recurrent urinary
tract infection (27), cholera (28) and infection with meningococcus
(29), pneumococcus (29), and haemophilus influenzae (30). The data
presented herein indicate an association between the non-secretor
status associated FUT2 genotype and CD.
[0021] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0022] The term "inflammatory bowel disease" or "IBD" refers to
gastrointestinal disorders including, but not limited to Crohn's
disease (CD), ulcerative colitis (UC), and indeterminate colitis
(IC). Inflammatory bowel diseases such as CD, UC, and IC are
distinguished from all other disorders, syndromes, and
abnormalities of the gastroenterological tract, including irritable
bowel syndrome (IBS).
[0023] "Risk variant" as used herein refers to genetic variants,
the presence of which correlates with an increase or decrease in
susceptibility to Crohn's disease. Risk variants of Crohn's disease
include, but are not limited to variants at the FUT2 genetic locus,
such as "haplotypes" and/or a set of single nucleotide
polymorphisms (SNPs) on a gene or chromatid that are statistically
associated. More preferably, risk variants can include, but are not
limited to rs602662, rs676388, rs485186, and rs504963.
[0024] "Treatment" or "treating," as used herein refer to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent, slow down and/or lessen the
disease even if the treatment is ultimately unsuccessful. Those in
need of treatment include those already with Crohn's disease as
well as those prone to have Crohn's disease or those in whom
Crohn's disease is to be prevented. For example, in Crohn's disease
treatment, a therapeutic agent may directly decrease the pathology
of IBD, or render the cells of the gastroenterological tract more
susceptible to treatment by other therapeutic agents.
[0025] As used herein, "diagnose" or "diagnosis" refers to
determining the nature or the identity of a condition or disease. A
diagnosis may be accompanied by a determination as to the severity
of the disease. Diagnosis as it relates to the present invention,
relates to the diagnosis of Crohn's disease.
[0026] As used herein, "prognostic" or "prognosis" refers to
predicting the probable course and outcome of IBD or the likelihood
of recovery from IBD. The prognosis can include the presence, the
outcome, or the aggressiveness of the disease.
[0027] As used herein, the term "biological sample" or "sample"
means any biological material obtained from an individual from
which nucleic acid molecules can be prepared. Examples of a
biological sample include, but are not limited to whole blood,
plasma, serum, saliva, cheek swab, urine, stool, or other bodily
fluid or tissue that contains nucleic acid.
[0028] In one embodiment, the present invention provides a method
of diagnosing susceptibility to Crohn's Disease in an individual,
relative to a healthy individual, by determining the presence or
absence of a risk variant at the FUT2 genetic locus, where the
presence of the risk variant at the FUT2 genetic locus is
indicative of susceptibility to Crohn's Disease in the individual.
In another embodiment, the risk variant comprises the SNP rs602662,
rs676388, rs485186, or rs504963. In one embodiment, the risk
variant can be at loci including, but are not limited to ASHL,
ARPC1A, RHOU, RBP1 and 2, TACR3, MMD2, NPSR1, ACER2, AP3D1, or
SPG20.
[0029] In one embodiment, the present invention provides a method
of treating Crohn's Disease by determining the presence of a risk
variant at the FUT2 genetic locus and treating the individual. The
risk variant comprises the SNP rs602662, rs676388, rs485186, and
rs504963. In one embodiment, the one or more risk variants can be
at loci including, but are not limited to ASHL, ARPC1A, RHOU, RBP1
and 2, TACR3, MMD2, NPSR1, ACER2, AP3D1, or SPG20.
[0030] In another embodiment, the present invention provides a
method of prognosing Crohn's Disease by determining the presence or
absence of one or more risk variants at the FUT2 genetic locus and
prognosing a complicated form of Crohn's Disease based on the
presence of the one or more risk variants at the FUT2 genetic
locus. The risk variant comprises the SNP rs602662, rs676388,
rs485186, and rs504963. In one embodiment, the one or more risk
variants can be at loci including, but are not limited to ASHL,
ARPC1A, RHOU, RBP1 and 2, TACR3, MMD2, NPSR1, ACER2, AP3D1, or
SPG20.
[0031] In one embodiment, the present invention provides a method
of diagnosing a high probability of developing Crohn's Disease in
an individual, relative to a healthy individual, by determining the
presence or absence of one or more risk variants at the FUT2
genetic locus, where the presence of the one or more risk variants
at the FUT2 genetic locus is indicative of a low probability of
developing Crohn's Disease in an individual. The risk variant
comprises the SNP rs602662, rs676388, rs485186, and rs504963. In
one embodiment, the one or more risk variants can be at loci
including, but are not limited to ASHL, ARPC1A, RHOU, RBP1 and 2,
TACR3, MMD2, NPSR1, ACER2, AP3D1, or SPG20.
[0032] In another embodiment, an individual with Crohn's disease
having one or more genetic risk variants at CD associated loci
specifically involved in the host-microbial interaction,
exemplified by, but not limited to, SPG20 and FUT2, is treated by
antibiotic and or probiotic based treatment therapies. In yet
another embodiment, the antibiotic and probiotic treatments are
administered as a preventative measure to individuals who have been
identified as having a higher than normal risk of developing CD,
based upon the presence of one or more genetic variants at CD
associated loci specifically involved in the host-microbial
interaction, exemplified by, but not limited to, SPG20 and
FUT2.
[0033] In another embodiment, the present invention provides a
method of prognosing Crohn's Disease by determining the presence or
absence of one or more risk variants of genetic loci at SPG20 and
FUT2, and prognosing pathogenesis, mediated in whole or in part by
host-microbial interaction, based on the presence of the one or
more risk variants at one or more of SPG20 and FUT2 genetic
loci.
[0034] A variety of methods can be used to determine the presence
or absence of a variant allele or haplotype. As an example,
enzymatic amplification of nucleic acid from an individual may be
used to obtain nucleic acid for subsequent analysis. The presence
or absence of a variant allele or haplotype may also be determined
directly from the individual's nucleic acid without enzymatic
amplification.
[0035] Analysis of the nucleic acid from an individual, whether
amplified or not, may be performed using any of various techniques.
Useful techniques include, without limitation, polymerase chain
reaction based analysis, sequence analysis and electrophoretic
analysis. As used herein, the term "nucleic acid" means a
polynucleotide such as a single or double-stranded DNA or RNA
molecule including, for example, genomic DNA, cDNA and mRNA. The
term nucleic acid encompasses nucleic acid molecules of both
natural and synthetic origin as well as molecules of linear,
circular or branched configuration representing either the sense or
antisense strand, or both, of a native nucleic acid molecule.
[0036] The presence or absence of a variant allele or haplotype may
involve amplification of an individual's nucleic acid by the
polymerase chain reaction. Use of the polymerase chain reaction for
the amplification of nucleic acids is well known in the art
(69).
[0037] A TaqmanB allelic discrimination assay available from
Applied Biosystems may be useful for determining the presence or
absence of a variant allele. In a TaqmanB allelic discrimination
assay, a specific, fluorescent, dye-labeled probe for each allele
is constructed. The probes contain different fluorescent reporter
dyes such as FAM and VICTM to differentiate the amplification of
each allele. In addition, each probe has a quencher dye at one end
which quenches fluorescence by fluorescence resonant energy
transfer (FRET). During PCR, each probe anneals specifically to
complementary sequences in the nucleic acid from the individual.
The 5' nuclease activity of Taq polymerase is used to cleave only
probe that hybridize to the allele. Cleavage separates the reporter
dye from the quencher dye, resulting in increased fluorescence by
the reporter dye. Thus, the fluorescence signal generated by PCR
amplification indicates which alleles are present in the sample.
Mismatches between a probe and allele reduce the efficiency of both
probe hybridization and cleavage by Taq polymerase, resulting in
little to no fluorescent signal. Improved specificity in allelic
discrimination assays can be achieved by conjugating a DNA minor
grove binder (MGB) group to a DNA probe as described, for example,
in Kutyavin et al., (67). Minor grove binders include, but are not
limited to, compounds such as dihydrocyclopyrroloindole tripeptide
(DPI,).
[0038] Sequence analysis also may also be useful for determining
the presence or absence of a variant allele or haplotype.
[0039] Restriction fragment length polymorphism (RFLP) analysis may
also be useful for determining the presence or absence of a
particular allele (68, 73). As used herein, restriction fragment
length polymorphism analysis is any method for distinguishing
genetic polymorphisms using a restriction enzyme, which is an
endonuclease that catalyzes the degradation of nucleic acid and
recognizes a specific base sequence, generally a palindrome or
inverted repeat. One skilled in the art understands that the use of
RFLP analysis depends upon an enzyme that can differentiate two
alleles at a polymorphic site.
[0040] Allele-specific oligonucleotide hybridization may also be
used to detect a disease-predisposing allele. Allele-specific
oligonucleotide hybridization is based on the use of a labeled
oligonucleotide probe having a sequence perfectly complementary,
for example, to the sequence encompassing a disease-predisposing
allele. Under appropriate conditions, the allele-specific probe
hybridizes to a nucleic acid containing the disease-predisposing
allele but does not hybridize to the one or more other alleles,
which have one or more nucleotide mismatches as compared to the
probe. If desired, a second allele-specific oligonucleotide probe
that matches an alternate allele also can be used. Similarly, the
technique of allele-specific oligonucleotide amplification can be
used to selectively amplify, for example, a disease-predisposing
allele by using an allele-specific oligonucleotide primer that is
perfectly complementary to the nucleotide sequence of the
disease-predisposing allele but which has one or more mismatches as
compared to other alleles (69). One skilled in the art understands
that the one or more nucleotide mismatches that distinguish between
the disease-predisposing allele and one or more other alleles are
preferably located in the center of an allele-specific
oligonucleotide primer to be used in allele-specific
oligonucleotide hybridization. In contrast, an allele-specific
oligonucleotide primer to be used in PCR amplification preferably
contains the one or more nucleotide mismatches that distinguish
between the disease-associated and other alleles at the 3' end of
the primer.
[0041] A heteroduplex mobility assay (HMA) is another well known
assay that may be used to detect a SNP or a haplotype. HMA is
useful for detecting the presence of a polymorphic sequence since a
DNA duplex carrying a mismatch has reduced mobility in a
polyacrylamide gel compared to the mobility of a perfectly
base-paired duplex (70, 71).
[0042] The technique of single strand conformational, polymorphism
(SSCP) also may be used to detect the presence or absence of a SNP
and/or a haplotype (72). This technique can be used to detect
mutations based on differences in the secondary structure of
single-strand DNA that produce an altered electrophoretic mobility
upon non-denaturing gel electrophoresis. Polymorphic fragments are
detected by comparison of the electrophoretic pattern of the test
fragment to corresponding standard fragments containing known
alleles.
[0043] Denaturing gradient gel electrophoresis (DGGE) also may be
used to detect a SNP and/or a haplotype. In DGGE, double-stranded
DNA is electrophoresed in a gel containing an increasing
concentration of denaturant; double-stranded fragments made up of
mismatched alleles have segments that melt more rapidly, causing
such fragments to migrate differently as compared to perfectly
complementary sequences (73).
[0044] Other molecular methods useful for determining the presence
or absence of a SNP and/or a haplotype are known in the art and
useful in the methods of the invention. Other well-known approaches
for determining the presence or absence of a SNP and/or a haplotype
include automated sequencing and RNAase mismatch techniques (74).
Furthermore, one skilled in the art understands that, where the
presence or absence of multiple alleles or haplotype(s) is to be
determined, individual alleles can be detected by any combination
of molecular methods (75). In addition, one skilled in the art
understands that multiple alleles can be detected in individual
reactions or in a single reaction (a "multiplex" assay). In view of
the above, one skilled in the art realizes that the methods of the
present invention for diagnosing or predicting susceptibility to or
protection against CD in an individual may be practiced using one
or any combination of the well known assays described above or
another art-recognized genetic assay.
[0045] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
EXAMPLES
[0046] The following examples are provided to better illustrate the
claimed invention and are not to be interpreted as limiting the
scope of the invention. To the extent that specific materials are
mentioned, it is merely for purposes of illustration and is not
intended to limit the invention. One skilled in the art may develop
equivalent means or reactants without the exercise of inventive
capacity and without departing from the scope of the invention.
Example 1
[0047] The discovery cohort used in the GWAS included 1096 Crohn's
Disease subjects and 3980 healthy population controls. The
replication cohort consisted of 1174 Caucasian CD cases and 357
Caucasian healthy controls; all independent of the cohort in the
GWAS. Cases were recruited from the Cedars-Sinai IBD Center and
Pediatric IBD department and were diagnosed with CD according to
standard clinical, radiological, endoscopic and histological
criteria. Controls for the GWAS were obtained from the
Cardiovascular Health Study (CHS), a population-based longitudinal
study of risk factors for cardiovascular disease and stroke in
adults 65 years of age or older, recruited at four field centers
(31). 5201 predominantly Caucasian individuals were recruited in
1989-1990 from random samples of Medicare eligibility lists,
followed by an additional 687 African-Americans recruited in
1992-1993 (total n=5888). Controls used in the replication study
were recruited through the IBD center (unrelated acquaintances and
spouses of cases with no personal or family history of IBD or
autoimmune disease) or recruited as part of the PARC project, a
pharmacogenetic study of statin response (32,33). All cases and
controls provided informed consent prior to study participation and
following approval of participating centers' institutional review
boards.
Example 2
[0048] All genotyping was performed at the Medical Genetics
Institute at Cedars-Sinai Medical Center using whole-genome
genotyping Infinium technology, following the manufacturer's
protocol (Illumina, San Diego, Calif.) (34, 35). Cases were
genotyped with either the Illumina Human 610Quad platform or the
Illumina Human 317Duo platform. Controls were genotyped with the
Illumina 370Duo platform. Samples with genotyping rates >98%
were retained in the analysis. In addition, case and control
cohorts were both investigated using Identity-By-Descent (Pi hat
scores >0.5 as detected in PLINK (36)) in order to identify
cryptic relatedness, and related individuals were excluded.
Following these QC steps, 1096 CD cases and 3694 controls were
included in the study. Single nucleotide polymorphisms (SNPs) were
excluded based on the following criteria: test of Hardy-Weinberg
Equilibrium p.ltoreq.10.sup.-3; SNP failure rate >10%; MAF
<5%; and SNPs not found in dbSNP Build 129. SNPs were also
examined in order to exclude case/control disparity in missingness
(PLINK (36)). 304,825 SNPs that passed QC criteria, and were
available in all datasets, were included in the logistic regression
association analysis. The 6 SNPs tested in the replication cohort
were genotyped using TaqMan.TM. assay according to the
manufacturer's instructions (Applied Biosystems, Foster City,
Calif.).
Example 3
[0049] Population structure was detected using Multidimensional
Scaling (MDS) (PLINK (36)). In total, 10 principal components (PC)
were calculated and plotted for graphical representation of
population substructure within the cohort. Subjects with a
PC1>0.025 represent African American subjects. To reduce false
positive discovery due to population substructure, and the
predominantly Caucasian make-up of the cases, these subjects were
excluded from downstream analysis. This resulted in 896 CD and 3204
control subjects being carried forward for association testing with
the CD phenotype using a logistic regression model in R (FIG. 2).
All 10 principal components were carried into association testing
as covariates. A logistic regression analysis correcting for
population substructure was used to test for association between
genotype and phenotype. Self reported ethnicity data was used to
confirm the identification of ethnicity based on cluster plots
(FIG. 2). The association of the FUT2 SNPs with CD in the
independent confirmation cohort was tested using logistic
regression (as implemented in R).
Example 4
[0050] A CD GWAS meta-analysis previously identified or confirmed
association with 30 loci and demonstrated nominal association with
a further 10 loci (13). The inventors confirmed association
(uncorrected p value <0.05 and association with the previously
identified risk allele) with 19 of these loci in the inventors'
GWAS (FIG. 3) and these loci served as internal controls for the
inventors' dataset. Three of these loci were from the nominally
replicated list of SNPs (rs4807569, 19p13; rs991804, CCL2, CCL7;
rs917997, IL18RAP) from the meta-analysis study, and the data
presented in FIG. 3 therefore provide further evidence of their
relevance in CD susceptibility. The IL18RAP association has
previously been confirmed (37). In this data set the inventors did
not demonstrate association (p<0.05) with CD and the other 21
loci identified in the GWAS meta-analysis including 10p11, 10q21,
12q12 (SLC2A13, LRRK2), 1p13 (PTPN22), 18p11 (PSMG2, PTPN2), 17q21
(ORMDL3), 13q14 (CCDC122), 9q32 (TNFSF15), 6p22 (CDKALI), 6q21
(PRDM1), 8q24, 1q23 (ITLN1, CD244,), 6p25(LYRM4), 2p16 (PUS10),
6p25 (SLC22A23), 6q25, 2p23 (GCKR), 7p12, 21q21, 21q22 and
18q11.
[0051] In addition, the inventors identified association between CD
and a number of novel loci (FIG. 4). These include genes involved
in tight junctions/epithelial integrity (ASHL, ARPC1A), Wnt and
JNK1 signaling (RHOU), dendritic cell function (RBP1 and 2),
Substance P signaling (TACR3), macrophage development (MMD2),
asthma susceptibility (NPSRJ) (38), integrin regulation (ACER2),
and NK T cell biology (AP3D1). The inventors also identified two CD
associated loci specifically involved in the host-microbial
interaction namely SPG20 (endosomal trafficking) and FUT2.
Example 5
[0052] From the novel associations, the inventors first chose FUT2
as the leading gene for independent replication given the
inventors' interest in the host-microbial interaction in CD
pathogenesis and FUT2's known association with a number of
infective processes. Furthermore FUT2 is located under a known peak
of linkage for CD on chromosome 19 (39) and there were 4 SNPs with
strong association to CD in the inventors' GWAS (FIGS. 5 and 6). In
addition to these 4 SNPs (rs504963--3'UTR, rs676388--3'UTR,
rs485186--synonymous exon 2 SNP and rs602662--Ser258Gly) identified
in the GWAS, the inventors also genotyped rs492602 (synonymous exon
2) and rs601338 (W143X, the common null allele in Caucasians
associated with the ABO non-secretory phenotype) in the independent
confirmatory cohort. The inventors were able to replicate the
initial association with the four SNPs from the discovery cohort,
as well as demonstrate association with the additional two SNPs,
including the allele for non-secretor status. Further evidence for
the association between this locus and CD susceptibility is
provided in the CD meta-analysis published by Barrett et al., (13)
in which all four of the originally identified SNPs are associated
with CD (FIG. 5). The 6 SNPs included in the replication study are
in strong linkage disequilibrium (FIG. 6).
Example 6
[0053] In this study the inventors confirmed association with a
number of known CD loci and provided further evidence for
association to CD with two other loci previously only nominally
associated with disease (19p13 and 17q12). The region on 19p13
contains SBNO2 and GPX4(glutathione peroxidase 4). Little is known
about SBNO4, while GPX4 is known to protect cells against oxidative
damage and may have a regulatory role in leukotriene biosynthesis
(40). The 17g12 locus is located in a cytokine gene cluster
containing the CCL2, CCL8, CCL11 and CCL7 genes. These genes encode
Cys-Cys cytokine genes which are involved in immunoregulatory and
inflammatory processes and are therefore attractive candidate genes
for CD susceptibility. This locus has previously been implicated in
susceptibility to asthma (41) and Mycobacterium susceptibility (42)
as well as with HIV progression (43).
[0054] Also disclosed herein, the inventors identified novel loci
associated with CD, most notably FUT2. The inventors provided
independent confirmation for association between FUT2 and CD in
both the inventors' own cohort, and in the meta-analysis published
by Barrett et al., (13). This cumulative data provides strong
evidence of the role of this locus in CD susceptibility. This gene
is of particular interest, as it potentially extends knowledge
regarding the scope of the host-microbial interaction in CD.
Previous genetic associations with CD have highlighted the role of
both the innate (11,12,14,15) and the adaptive immune systems'
(44,45) interaction with the microbiome. The data presented herein
extend this interaction to the mucus layer of the GI tract. FUT2
encodes the secretor type .alpha. (1,2) fucosyltransferase (also
known as the Se enzyme) that is responsible for regulating the
secretion of the ABO antigens in both the digestive mucosa and
secretory glands. Approximately 20% of individuals are
non-secretors who fail to express ABO antigens in both the GI tract
and saliva (17). The prevalence of the non-secretor status (Sc-) is
similar between populations (46) although the point mutations that
lead to Se- differ. The dominant non-secretor polymorphism in
caucasians is the Trp143Ter (W143X) (17) and it is this
polymorphism that is implicated in CD in the replication
cohort.
[0055] Pathogens utilize host cell surface molecules including
oligosaccharides (synthesized by glycosyltransferases) for
invasion. It is likely that the high prevalence of non-secretor
phenotypes in the population occurs due to the absence of
particular carbohydrate molecules in the mucosa, and this may have
conferred some historical protection to infection as demonstrated
with non-secretor status and protection from Helicobacter Pylori
infection (18). Lactobacilli, a known commensal bacteria, bind to
the precursor glycolipid GA1, implying a role of the GI mucosal
glycolipid profile in the adherence of commensal and `beneficial`
bacteria, in addition to pathogenic organisms (47). Furthermore
Lactobacilli can also displace pathogens such as Clostridium from
mucus (48) and inhibit the Shigella-host interaction (49).
Commensal bacteria likely induce glycolipid expression, as the
fucosylglycolipid FGA1 is found in the small bowel of
conventionally bred mice but not in germ-free mice (50).
Furthermore FGA1 expression is induced by administration of
microbes (51), and FUT2 transcripts in the ileum were induced in
germ free mice 48 hours after administration of feces from
conventionally bred mice (52). Fut2-null mice do not express the
fucosylglycolipid FGA1 in the cecum and colon, whereas normal mice
do (50). In the mammalian gut, blocking the CRK and INK pathways
inhibits the ability of bacterial colonization to induce
fucosyltransferase activity and FUT2 mRNA expression, both of which
are hallmarks of the adult mammalian colon (53). Commensal bacteria
and probiotics may exert their protective effects through
preventing adherence or even displacing pathogenic bacteria, thus
emphasizing the potential role of FUT2 and non-secretor status on
gastrointestinal bacterial profile (54). It is likely that Se-
individuals may thus have a disrupted immunogenic/homeostatic
equilibrium that makes them more susceptible to the development of
chronic mucosal inflammation, and changes in the microflora of IBD
patients have been well-documented (55). There are some data to
support this concept, as Fut2 null mice display increased
susceptibility to experimental yeast vaginitis and cervical mucins
containing Fut2 are partly protected from induced vaginal
candidiasis (56).
[0056] Although FUT2 is a strong candidate gene for CD
susceptibility, given its tissue expression and its influence on
the GI bacterial profile, the associations identified in FUT2 may
reflect association with other genetic variants at this locus that
are in linkage disequilibrium with these SNPs. The inventors
therefore explored the LD pattern at this locus using the latest
version of HapMap (57) and identified that LD (defined as
D'>0.80) extends into neighboring genes, including interesting
candidate genes that are also potentially involved in the
host-bacterial interaction such as FUT1
(alpha-1-2-fucosyltransferase 1--FUT, genetic variation in pigs is
associated with alterations in E. Coli adherence (58)) and RASIPI
(RAS interacting protein 1--a RAS effector localized to the Golgi
membranes) as well as DBP (D-site of albumin promoter-binding
protein) and FGF21 (fibroblast growth factor 21--involved in
insulin sensitivity, adipocyte function and growth hormone
signalling (59,60)). The inventors believe that FUT2 is an
attractive candidate gene at this locus, and have demonstrated
association with a variant with a known consequence on gene
expression.
[0057] In addition, the inventors have identified some novel loci
for further investigation, including genes involved in tight
junctions, Substance P signaling, macrophage development, dendritic
cell function and NK T cell function.
[0058] The data disclosed herein provide strong evidence that
non-secretor status increases CD susceptibility. The non-secretor
variants from other ethnic groups have been well documented, and
studies of these variants within the relevant IBD populations will
help elucidate the exact role of FUT2 in CD susceptibility. Studies
on the effect of FUT2 on clinical and serological phenotype, and
particular its role on the microbiome of non-secretor individuals,
may help investigators understand further the variation seen in
commensal bacteria in individuals with CD, and also further
determine those CD patients who might most benefit from probiotic
or antibiotic based therapies for prevention and treatment of
CD.
[0059] While particular embodiments of the present invention have
been shown and described, it will be obvious to those skilled in
the art that, based upon the teachings herein, changes and
modifications may be made without departing from this invention and
its broader aspects and, therefore, the appended claims are to
encompass within their scope all such changes and modifications as
are within the true spirit and scope of this invention.
[0060] Many modifications and variations of the invention as
hereinbefore set forth can be made without departing from the
spirit and scope thereof and therefore only such limitations should
be imposed as are indicated by the appended claims.
[0061] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
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